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Introduction

Hepatocellular carcinoma (HCC) was the seventh leading type of cancer and the fourth leading cause of cancer-related death worldwide in 2017 (1,2). Liver cancer has a poor prognosis, with a ratio of mortality to incidence of 0.93 worldwide (2). Surgical resection, transplantation and percutaneous ablation are clinically available for the treatment of early-stage HCC (3,4). However, resistance to chemotherapy or radiotherapy has resulted in an absence of effective therapeutic options and a poor prognosis, especially for patients with advanced HCC (5). Therefore, it is important to develop novel treatments or to reverse chemoresistance to improve the outcomes of patients with advanced HCC.

Eukaryotic translation initiation factor 5A (eIF5A or eIF5A1) is highly conserved throughout eukaryotic evolution and serves roles in mRNA translation, cellular proliferation, differentiation and inflammation (6). eIF5A and its homolog eIF5A2, a 17-kDa protein comprising 153 amino acids, are the only known proteins that contain a unique amino acid residue, hypusine, which is crucial for their function to maintain polyamine homeostasis (6,7). eIF5A2 is of particular interest since it is rare in the majority of normal tissues but abundant in the testes, parts of the brain and several types of malignant tissues including bladder and colorectal cancer as well as hepatocellular carcinoma (8,9). eIF5A2 has been proposed to be an oncogene, and overexpression of eIF5A2 has been demonstrated to promote cell aggressiveness in colorectal carcinoma (10), bladder cancer (11), melanoma (12) and gastric cancer (13). Furthermore, inhibition of eIF5A2 by N1-guanyl-1, 7-diaminoheptane, an inhibitor of deoxyhypusine synthase, has been demonstrated to enhance the chemotherapeutic effects of gemcitabine in pancreatic ductal adenocarcinoma cells (14) and that of doxorubicin in bladder cancer cells (11). Although a number of studies have been performed to understand the relationship between eIF5A2 and chemosensitivity (11,14), the mechanism underlying eIF5A2 and chemosensitivity in HCC remains unclear.

Autophagy, a lysosomal degradation pathway for providing energy and macromolecular precursors (15), has also been implicated in the development of chemoresistance. When cells encounter environmental stressors, such as nutrient starvation and pathogen infection, autophagy is induced to digest and recycle cellular proteins and organelles to sustain cellular metabolism, or to eliminate pathogens and apoptotic cells (16,17). Autophagy is also coordinated with the ubiquitin-proteasome system to remove polyubiquitinated and aggregated proteins (18). Therefore, autophagy may promote cell survival and contribute to chemoresistance. A number of studies have demonstrated that inhibition of autophagy overcomes chemoresistance in prostate cancer, leukemia and bladder cancer (19-21). The induction of autophagy has also been reported to cause a form of programmed cell death termed autophagic cell death (22-24). Furthermore, Segala et al (25) have reported that dendrogenin A, a cholesterol metabolite, directly controls a nuclear receptor to trigger lethal autophagy in melanoma. Autophagy has been identified as a cytoprotective mechanism in gastric carcinoma, leukemia and squamous cell carcinoma (26-28). In addition, autophagy serves a cytocidal role in breast and colorectal cancer (29,30). However, the role of autophagy in the chemoresistance or chemosensitivity in HCC remains controversial.

The present study aimed to determine the potential roles of eIF5A2 in doxorubicin sensitivity and to investigate the effects of autophagy during this process.

Materials and methods

Ethics statement

The present study was approved by the Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (approval no. 2018-238; Hangzhou, China). All samples were anonymously coded in accordance with local ethical guidelines (based on the Declaration of Helsinki), and written informed consent was obtained from all patients. All animals used received appropriate care according to the Institutional Animal Care and Use Committee at the Second Affiliated Hospital of Zhejiang University School of Medicine (approval no. 2018-311). All efforts were made to minimize animal suffering.

Cell lines and culture

The human hepatocellular carcinoma cell lines SNU449, SNU387 and Huh7 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai Institute of Cell Biology, Chinese Academy of Sciences. SNU449 and SNU387 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.). Huh7 cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 mg/ml streptomycin, and all cells were maintained at 37°C in a humidified incubator with 5% CO2.

Antibodies and reagents

Anti-LC3B (1:1,000; cat. no. 3868S), SQSTM1/p62 (1:1,000; cat. no. 8025S), Beclin 1 (1:1,000, cat. no. 3495P), HRP-conjugated anti-mouse IgG (1:2,000; cat. no. 7076S) and HRP-conjugated anti-rabbit IgG (1:2,000; cat. no. 7074S) antibodies were purchased from Cell Signaling Technology, Inc. The anti-eIF5A2 (1:1,000; cat. no. ab126735) and anti-KI67 (1:200; cat. no. ab16667) antibodies were obtained from Abcam, and the anti-β-actin (1:1,000; cat. no. 66009-1-ig) antibody was from ProteinTech Group, Inc..

The eIF5A2 small interfering RNA (siRNA) and negative control siRNA were synthesized by Shanghai GenePharma Co., Ltd. The PT3-EF1a-eIF5A2-flag and PT3-EF1a plasmids were purchased from Wuhan Yuling Biological Technology Co., Ltd. Cell Counting Kit-8 (CCK-8; cat. no. AD10) was obtained from Dojindo Molecular Technologies, Inc. The 5-ethynyl-2′-deoxyuridine (EdU) kit (cat. no. A10044) and Lipofectamine® 2000 Transfection Reagent (cat. no. 11668019) were purchased from Invitrogen; Thermo Fisher Scientific, Inc. The autophagy inhibitor chloroquine (CQ; cat. no. C6628) and the mTOR inhibitor rapamycin (Rapa; cat. no. V900930) were obtained from Sigma-Aldrich; Merck KGaA. Doxorubicin (cat. no. S1208) was purchased from Selleck Chemicals. Monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP)-LC3 adeno-associated virus (AAV) was obtained from Hanbio Biotechnology Co., Ltd. 2′-O-methoxyethyl (2′-Ome)- and 5′-cholesterol (5′-chol)-modified eIF5A2 siRNA was chemically synthesized by Guangzhou RiboBio Co., Ltd.

Survival analysis

The tissue microarray containing 90 paired HCC and adjacent tissues was obtained from Shanghai Xinchao Biological Technology Co. Ltd. (cat. no. HLiv-HCC180Sur-04). The clinicopathological information about patient age, sex, tumor stage and survival was provided by Shanghai Xinchao Biological Technology Co. Ltd. The follow-up period ranged between 1 and 6 years. Immunohistochemistry (IHC) staining of eIF5A2 was performed as follows: The tissue microarray was incubated with 3% H2O2 for 10 min at room temperature, followed by antigen retrieval in Tris-EDTA buffer containing 0.05% Tween 20 (pH 9.0) in a water bath at 95-100°C for 15 min, incubation with 10% FBS in PBS for 30 min at room temperature, and further incubation with an anti-eIF5A2 (1:100; cat. no. ab126735; Abcam) antibody at 4°C overnight. The tissues were subsequently incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:200; cat. no. ab205718; Abcam) at room temperature for 30 min, followed by incubation with DAB (cat. no. ab64238; Abcam) at room temperature for 1-2 min according to the manufacturer's instructions and counterstaining with hematoxylin. The stained tissues were dehydrated and stabilize with mounting medium (cat. no. ab64230; Abcam). Images (magnification, ×200) were captured using AperioImageScope software 11.1.2.752 (Leica Biosystems), and 5-10 fields of view per sample were analyzed. IHC analysis of the paired HCC and adjacent tissues in the microarray was used to evaluate eIF5A2 staining intensity and relative expression levels in tumors and adjacent tissues. Images (magnification, ×200) were captured using Aperio ImageScope software 11.1.2.752 (Leica Biosystems), and 5-10 fields of view per sample were analyzed. The tissue scoring was confirmed by two indepen-dent pathologists who were blinded to the grouping. eIF5A2 staining score was based on staining intensity as follows: 0, none; 1, equivocal/uninterpretable; 2, weak; and 3, intermediate-strong. The proportion score was based on expression rate as follows: 0, none; 1, 1-25%; 2, 26-50%; 3, 51-75%; and 4, 76-100%. A composite score was obtained by multiplying the intensity score by the proportion score. A composite score <4 was considered low expression, and a score ≥4 was considered high expression. Long-term follow-up was obtained through tumor registries and telephone interviews. The survival curve was drawn by Kaplan-Meier method, and the difference was evaluated by log-rank test.

Patient samples

A total of 30 paired tumor and adjacent tissues (≥3 cm from the tumor margin) were collected from patients with HCC (mean age, 56 years; range, 38-78 years; 21 male and 9 female patients) undergoing surgery at the Second Affiliated Hospital of Zhejiang University School of Medicine between January and December 2018. The histological diagnosis of HCC was confirmed by two independent pathologists, and the samples were used for reverse transcription-quantitative PCR (RT-qPCR) and mouse model establishment.

RNA isolation

A total of 50-100 mg of HCC or adjacent tissue was homogenized in 1 ml TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) using a power homogenizer. For cells, 1 ml TRIzol® reagent was added to a culture dish to lyse the cells directly. The homogenized samples were incubated for 5 min at room temperature, and 0.2 ml chloroform per 1 ml of TRIzol reagent was added, followed by centrifugation of the samples at 12,000 × g for 15 min at 4°C. Subsequently, the upper aqueous phase was transferred into fresh tubes, and 0.5 ml isopropyl alcohol was added, followed by centrifugation at 12,000 × g at 4°C for 10 min. The supernatant was removed, and the RNA pellet was washed with 75% ethanol. Finally, the RNA was dissolved in DEPC-treated water at room temperature.

RT-qPCR

Following total RNA extraction, reverse transcription of mRNA was performed using a PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara Bio, Inc.) according to the manufacturer's instructions. The mRNA levels of eIF5A2 and β-actin were quantified by qPCR using specific primers and a TB Green qPCR Master Mix (Takara Bio, Inc.) on a LightCycler® 480 II instrument (Roche Diagnostics). The thermocycling conditions were as follows: 95°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C for 30 sec; and 50°C for 30 sec. The primer sequences were as follows: eIF5A2 forward, 5′-TAT GCA GTG CTC GGC CTT G-3′ and reverse, 5′-TTG GAA CAT CCA TGT TGT GAG TAG A-3′; and β-actin forward, 5′-TTC CAG CCT TCC TTC CTG-3′ and reverse, 5′-CTT TGC GGA TGT CCA CGT-3′. Relative changes in the mRNA expression levels were analyzed using the 2−ΔΔCq method (31). All experiments were carried out in triplicate and independently repeated three times.

Cell viability assay

Tumor cells were seeded into 96-well plates at 3,000 cells/well. Following adherence, the medium was replaced with medium containing 0, 0.125, 0.25, 0.5, 1, 2 or 5 µg/ml doxorubicin for 48 h at 37°C. Subsequently, CCK-8 solution was added into the wells (1:1,000) for 2 h at 37°C according to the manufacturer's instructions, and cell viability was determined at 450 nm using an Epoch Microplate Spectrophotometer (BioTek Instruments, Inc.). For the co-treatment with CQ or rapamycin, SNU449 cells were pretreated with 10 µM CQ (dissolved in ddH2O) or 100 nM rapamycin (dissolved in DMSO) for 6 h and subsequently treated with eIF5A2 siRNA and doxorubicin.

In vivo analysis

A total of 20 male athymic BALB/c mice (age, 4-5 weeks; weight, 15-18 g) were obtained from the Model Animal Research Center of Nanjing University, Nanjing, China. The mice were housed in specific pathogen-free barrier facilities in a temperature-controlled room (24°C) with a 12 h light-dark cycle and 35-40% relative humidity, and were permitted free access to food and drinking water. The body weight of each mouse was measured every 2 days. The mice were humanely sacrificed by cervical dislocation at the study endpoint.

Patient-derived xenograft (PDX) tumor tissues were used to establish the tumor model. Patient tumor in which eIF5A2 was detected by RT-qPCR was used to establishing the mouse model. The resected tumor was cut into 1 mm3 fragments and inoculated subcutaneously into the flanks of the mice (one fragment per mouse). After 3 weeks, when the tumor volumes reached 50-100 mm3, the mice were randomly divided into the following groups (n=5 mice/group): i) Control (saline); ii) doxorubicin; iii) eIF5A2 siRNA; and iv) eIF5A2 siRNA combined with doxorubicin. Saline and doxorubicin (2 mg/kg) were injected via the tail vein every 2 days. eIF5A2 siRNA (1 nmol/mouse) was administered via intratumoral injection every 2 days. To maintain the the stability of eIF5A2 siRNA in vivo, a 2′-OMe and 5′-chol modified siRNA was used. The 2′-OMe modification inhibits the ability of RNase H to cleave the bound sense RNA strand within the heteroduplex formed between the nucleic acid and the target RNA (32), and the 5′-chol modification alters protein binding in serum, extending the circulation time, and facilitates direct cellular uptake (33). The body weights and tumor dimensions were measured every other day. The tumor volume was calculated using the modified ellipsoid formula: Volume=1/2 × (length × width2). After 14 days, all mice were sacrificed, and the blood, liver, kidney and tumor tissues were harvested. The tissues were fixed with 4% formaldehyde for 48 h at 25°C, and the liver, kidney and a part of each tumor were used for IHC. The remaining tumor tissues used for protein and mRNA expression analysis of eIF5A2 and autophagy-related genes.

IHC analysis and TUNEL assay

After the mice were sacrificed, the tumor tissues were dissected and fixed in formaldehyde. Following paraffin embedding, sectioning (5 µm), deparaffinization, incubation in 3% H2O2 for 10 min at room temperature, antigen retrieval by boiling in 0.01 M sodium citrate buffer (pH 6.0) for 15 min, the tissues were blocked with 10% FBS in PBS for 30 min at room temperature and stained with an anti-ki67 antibody (1:200) at 4°C overnight. Subsequently, the sections were incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:200) for 30 min at room temperature, incubated with DAB at for 1-2 min room temperature, counterstained with hematoxylin, dehydrated and stabilized with mounting medium (cat. no. ab64230; Abcam). For TUNEL assay, an in situ Cell Death Detection kit (Roche Molecular Diagnostics) was used to detect apoptosis according to the manufacturer′s instructions following deparaffinization. The sections were counterstained with hematoxylin for 3 min at room temperature following dehydration and stabilizing with mounting medium. The slides were visualized under a Leica DM300 microscope (magnification, ×200; Leica Microsystems GmbH), and 5-10 fields of view per sample were quantified using Leica Application Suite X software 3.0.0.15697 (Leica Microsystems GmbH).

Transfection

HCC cells (2×105) in the logarithmic phase of growth were seeded into 6-well plates and transfected with eIF5A2 siRNA (50 pmol), control siRNA (50 pmol), pT3-EF1a-eIF5A2-flag plasmid (2 µg) or pT3-EF1a plasmid (2 µg) when the cells reached a density of 50-60% using Lipofectamine® 2000 according to the manufacturer's instructions. The transfection medium (Opti-MEM; Gibco; Thermo Fisher Scientific, Inc.) was replaced with complete medium following 6-h incubation at 37°C, and the cells were harvested after 48 h. The sequences of siRNAs were as follows: Negative control-homo sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and anti-sense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′; eIF5A2-homo sense, 5′-GCA GAC GAA AUU GAU UUC ATT-3′ and anti-sense, 5′-UGA AAU CAA UUU CGU CUG CTT-3′; and Beclin 1-homo sense, 5′-GUA CCG ACU UGU UCC CUA UTT-3′ and anti-sense, 5′-AUA GGG AAC AAG UCG GUA CTT-3′.

Western blotting analysis

Cells were harvested in cell lysis buffer (Cell Signaling Technology, Inc.) containing protease inhibitors (Sigma-Aldrich; Merck KGaA) following treatment with eIF5A2 siRNA and doxorubicin for 48 h. The protein concentration was determined by BCA assay (Applygen Technologies, Inc.). A total of 10 µg protein was loaded per lane, separated by 15% SDS-PAGE and transferred to 0.45-µm polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with TBS-Tween-20 (0.1% Tween-20) containing 0.5% BSA (cat. no. 4240GR100; NeoFroxx GmbH) and incubated with the aforementioned primary antibodies at 4°C overnight. The membranes were washed with TBS-0.1% Tween-20 three times. Following incubation with anti-mouse IgG or anti-rabbit IgG secondary antibodies at 4°C for 2 h, protein expression was detected by chemiluminescence with an EZ-ECL Chemiluminescence Detection Kit (Biological Industries).

EdU assay

Tumor cells were seeded in a 96-well plate at 3,000 cells/well and incubated with medium (RPMI-1640 medium for SNU449 and DMEM for Huh7) containing doxorubicin at the IC50 concentration for 48 h. Prior to fixation with 4% paraformaldehyde at 25°C for 15 min, the cells were incubated with 10 µM EdU at 37°C for 2 h. Next, membrane permeabilization for 15 min, EdU incubation for 30 min and Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) staining for 15 min were performed to stain EdU and the cell nuclei at 25°C, respectively. Images were captured under na Olympus IX73 fluorescence microscope (magnification, ×200; Olympus Corporation), and 5-10 fields of view per sample were analyzed.

Transmission electron microscopy (TEM) analysis

Following transfection with eIF5A2 siRNA for 48 h, SNU449 cells were fixed with glutaraldehyde at 4°C for 16 h, followed by treatment with 1% osmic acid at 25°C for >2 h, then rinsed with 0.1 M phosphate buffer, dehydrated sequentially in 50, 70 and 90% ethanol, 90% ethanol plus 90% acetone, 90 and 100% acetone for 15 min per step, and then embedded in Epon 812 embedding medium (Structure Probe, Inc.) according to the manufacturer's instructions. The samples were sectioned at 50-60 nm, stained with 3% uranyl acetate plus lead citrate, and images were captured using a transmission electron microscope (magnification, ×25,000), and five fields of view per sample were analyzed.

Confocal microscopy

SNU449 cells were seeded into confocal dishes at 3×105 cells/well. Cells were treated with RPMI-1640 medium containing the mRFP-GFP-LC3 AAV at 37°C for 6 h, incubated with eIF5A2 siRNA and doxorubicin at 37°C for 48 h and fixed with 4% paraformaldehyde for 15 min at 25°C. Images were captured using an Olympus IX81-FV1000 confocal microscope (magnification, ×1,600; Olympus Corporation), and 5-10 fields of view of per sample were analyzed.

Apoptosis assay

Apoptosis was detected using Annexin V, FITC Apoptosis Detection kit (Dojindo Molecular Technologies, Inc.) After eIF5A2 siRNA and doxorubicin treatment for 48 h, 1×106 SNU449 cells were harvested by trypsinization and collected by centrifugation (300 × g at 4° for 3 min), resuspended in 100 µl binding buffer, stained with 5 µl annexin V-FITC and 5 µl propidium iodide (PI) at room temperature in the dark for 15 min according to the manufacturer's instructions, and then analyzed using a BD FACScanto II flow cytometer with BD FACSDiva software v8.0.1 (both from BD Biosciences).

Lactate dehydrogenase (LDH) release assay

HCC cells were seeded into a 96-well plate at 3,000 cells/well and administered eIF5A2 siRNA, eIF5A2 plasmid, CQ and rapamycin, alone or in combination at 37°C for 48 h. The culture medium was collected by centrifugation at 300 × g for 3 min (4°C) and transferred to a new 96-well plate, and the release of LDH was measured using a Cytotoxicity LDH Assay kit at 25°C (Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. LDH release was detected at 490 nm using an Epoch Microplate Spectrophotometer.

Cell death assay

SNU449 cells (2×105) in the logarithmic phase of growth were seeded in 6-well plates. After cells were administered eIF5A2 siRNA and Beclin 1 siRNA, alone or in combination at 37°C for 48 h, cells were harvested by trypsinization and collected by centrifugation at 300 × g for 3 min, resuspended in trypan blue solution (T10282, Thermo Fisher Scientific, Inc.) and counted using a Countess II Automated Cell Counter with Countess II/II FL software v1.0.247 (both from Thermo Fisher Scientific, Inc.). The cell counter automatically quantified the trypan blue-positive dead cells and the trypan blue-negative living cells.

Calcein-AM/PI staining

SNU449 cells were seeded in a 96-well plate at 3,000 cells/well and administered eIF5A2 siRNA, eIF5A2 plasmid, CQ and rapamycin, alone or in combination at 37°C for 48 h. The cells were then cultured with 1 µM calcein-AM and 3 µM PI (Cell Signaling Technology, Inc.) at 25°C for 30 min. Subsequently, images were captured using an Olympus IX73 fluorescence microscope (magnification, ×100), and five fields of view per were analyzed.

Statistical analysis

Data are presented as the mean ± SD from ≥3 independent replicates. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc.). Unpaired two-tailed Student's t-test was used to analyze differences between two groups, and one-way ANOVA with Tukey's post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Results

eIF5A2 expression is negatively associated with the prognosis of patients with HCC

To investigate the role of eIF5A2 in HCC, a tissue microarray was used. Immunohistochemical staining of eIF5A2 protein in paired tumor and adjacent tissues from 90 patients with HCC revealed that the staining intensity of eIF5A2 was higher in tumor tissues compared with that in normal tissues (Fig. 1A and B). Based on the aberrant eIF5A2 expression, survival analysis revealed that high expression levels of eIF5A2 were associated with a shorter overall survival time of patients with HCC (Fig. 1C). In addition, eIF5A2 mRNA expression was detected in 30 pairs of tumors and adjacent tissues from patients with HCC. The tumor tissues exhibited higher expression levels of eIF5A2 compared with those in the adjacent normal tissues (Fig. 1D). These results suggested that eIF5A2 mRNA and protein expression levels were upregulated in HCC compared with those in normal tissues, and were associated with a poor prognosis.

Figure 1 eIF5A2 expression is negatively associated with prognosis of patients with liver cancer. (A) Immunohistochemical staining of eIF5A2 protein in paired tumor and adjacent tissues. (B) Composite staining scores for eIF5A2 protein from 90 paired tumor and adjacent tissues. (C) Overall survival of 90 patients with HCC. (D) RT-qPCR analysis of eIF5A2 mRNA in paired tumor and adjacent tissues from patients with HCC (n=30). (E) HCC cells were treated with the indicated doses of doxorubicin for 48 h. Cell viability was determined by the Cell Counting Kit-8 assay. (F) RT-qPCR analysis of eIF5A2 mRNA in HCC cells. (G) Association between eIF5A2 expression and IC50 values of doxorubicin in HCC cells. *P<0.05 and **P<0.01. eIF5A2, eukaryotic translation initiation factor 5A2; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

To characterize the relationship between eIF5A2 and doxorubicin sensitivity, three HCC cell lines (SNU449, SNU387 and Huh7) were treated with 0.125-5 µg/ml doxorubicin. Sensitivity to doxorubicin was measured after 48-h treatment by CCK-8 assay. As demonstrated in Fig. 1E, among the tested cell lines, SNU449 was the most resistant to doxorubicin, followed by SNU387 and Huh7. Basal levels of eIF5A2 mRNA in the three cell lines were also assessed by RT-PCR (Fig. 1F). SNU449 had the highest expression of eIF5A2 while Huh7 had the lowest. Notably, the IC50 values for doxorubicin appeared to be associated with eIF5A2 expression (Fig. 1G). Collectively, these results suggested that eIF5A2 may serve as a biomarker of HCC and may be involved in doxorubicin sensitivity.

eIF5A2 silencing improves doxorubicin chemotherapy in vivo

To investigate the role of eIF5A2 in chemosensitivity, three specific siRNAs were designed to silence the expression of eIF5A2 (Fig. S1), and the most efficient one (sieIF5A2-3, further referred to as eIF5A2 siRNA) was selected for further experiments.

To further determine the effects of eIF5A2 on doxorubicin sensitivity, a patient-derived xenograft (PDX) mouse model was established. An HCC tumor with high expression levels of eIF5A2 (Fig. S2) was subcutaneously injected into mice. Following 14-day treatment, both doxorubicin administration and eIF5A2 siRNA injection were observed to inhibit tumor growth (Fig. 2A-C). Notably, co-administration of eIF5A2 siRNA markedly enhanced the therapeutic effects of doxorubicin on tumor suppression (Fig. 2A-C). Doxorubicin and eIF5A2 siRNA exhibited no significant effects on the mouse body weight (Fig. 2C). In addition, the combination of doxorubicin and eIF5A2 siRNA inhibited the proliferation of tumor cells (as demonstrated by IHC staining of ki67) and increased the apoptotic rate (analyzed by TUNEL assay) compared with those in the other three groups (Fig. 2D), suggesting that co-treatment with of eIF5A2 siRNA may improve the therapeutic efficiency of doxorubicin in vivo.

Figure 2 eIF5A2 silencing improves the efficacy of Dox chemotherapy in vivo. (A) Athymic nude mice (n=5 mice/group) engrafted with patient-derived xenograft tumor tissues were treated with saline, doxorubicin, sieIF5A2 or sieIF5A2 plus Dox. Xenograft tumors of hepatocellular carcinoma were imaged after 2-week treatment. (B) Tumor weight was assessed at the end of the experiments. (C) Tumor volumes and mouse body weights were monitored over time. (D) Immunohistochemical analysis of the expression of ki67 and TUNEL staining analysis in xenograft tumors dissected from mice of from the four experimental groups. The frequency of ki67 positive cells and TUNEL-positive cells was quantified. *P<0.05, **P<0.01 and ***P<0.001. eIF5A2, eukaryotic translation initiation factor 5A2; si, small interfering RNA; NC, negative control; Dox, doxorubicin.

eIF5A2 is involved in regulating autophagy and chemosensitivity of HCC cells

To further determine the tumor-suppressing effects of eIF5A2 siRNA observed in the in vivo experiment, SNU449 and Huh7 cells were transfected with eIF5A2 siRNA or an eIF5A2-overexpressing plasmid, respectively, and doxorubicin sensitivity was evaluated using the CCK-8 assay. The results demonstrated that silencing of eIF5A2 enhanced the effects of doxorubicin on cell viability compared with those in the negative control group, whereas eIF5A2 upregulation impaired the cytotoxic effects of doxorubicin (Fig. 3A and B). In addition, doxorubicin treatment alone notably reduced the proliferation rate in SNU449 and Huh7 cells according to the results of the EdU incorporation assay, and transfection with eIF5A2 siRNA and doxorubicin treatment exhibited additive antiproliferative effects (Fig. 3C). By contrast, eIF5A2 overexpression promoted HCC cell proliferation of doxorubicin-treated cells. These results demonstrated that eIF5A2 silencing enhanced doxorubicin efficacy in vitro.

Figure 3 eIF5A2 is involved in the regulation of autophagy and chemosensitivity in HCC cells. (A) SNU449 cells were treated with indicated concentrations of Dox for 48 h following transfection with NC siRNA or sieIF5A2. Huh7 cells were treated with indicated concentrations of Dox for 48 h following transfection with an empty plasmid or OE eIF5A2. Cell viability was measured by the Cell Counting Kit-8 assay. (B) Western blotting analysis of eIF5A2 protein expression levels in HCC cells transfected with sieIF5A2 or OE eIF5A2. (C) HCC cells were treated with or without Dox for 48 h in the presence or absence of sieIF5A2 or OE eIF5A2. The cell proliferation rate was evaluated by EdU incorporation assay, and the frequency of EdU positive cells was quantified. (D) Autophagosomes were visualized in SNU449 cells treated with NC siRNA or sieIF5A2 by transmission electron microscopy. (E) SNU449 cells were infected with the mRFP-GFP-LC3 adenovirus and subsequently transfected with NC siRNA or sieIF5A2 and visualized by confocal microscopy. The numbers of GFP+/mRFP+-LC3 (yellow, autophagosomes) and GFP-/mRFP+-LC3 (red, autolysosomes) dots were counted and analyzed. *P<0.05 and **P<0.01. eIF5A2, eukaryotic translation initiation factor 5A2; EdU, 5-ethynyl-2′-deoxyuridine; HCC, hepatocellular carcinoma; siRNA, small interfering RNA; NC, negative control; sieIF5A2, siRNA against eIF5A2; OE, overexpression vector; mRFP, monomeric red fluorescent protein; GFP, green fluorescent protein; Dox, doxorubicin.

eIF5A2 silencing also resulted in increased numbers of autophagosomes observed by TEM compared with those in the negative control group (Fig. 3D). In order to validate the appearance of autophagic characteristics, the cells were infected with an AAV expressing an mRFP-GFP-LC3 reporter protein for monitoring the autophagic flux (Fig. 3E). The results demonstrated that eIF5A2 knockdown induced an increase in the numbers of in autophagosomes and autoly-sosomes compared with those in the negative control group, suggesting that eIF5A2 siRNA-induced autophagy may be involved in doxorubicin sensitivity.

Silencing of eIF5A2 induces sensitivity to doxorubicin in HCC cells by triggering lethal autophagy

To determine the role of autophagy triggered by eIF5A2 siRNA in doxorubicin treatment, the autophagic flux of cells treated with doxorubicin and eIF5A2 siRNA alone or in combination was visualized. Knockdown efficiency of eIF5A2 siRNA was validated by western blotting (Fig. 4A). Doxorubicin treatment induced autophagic flux in SNU499 cells compared with that in the control group (Fig. 4B and C). In addition, combined eIF5A2 siRNA and doxorubicin treatment further reinforced the autophagic activity, with an increased number of autophagosomes compared with that observed in cells treated with doxorubicin alone (Fig. 4B and C). The protein expression levels of LC-3 I/II, Beclin 1 and p62 were evaluated by western blotting. The results demonstrated that eIF5A2 silencing markedly promoted the conversion of LC3I to LC3II, the formation of Beclin1 and the degradation of p62 compared with those in cells treated with doxorubicin alone (Fig. 4C), confirming that eIF5A2 siRNA promoted autophagy induced by doxorubicin. In addition, an increase of LDH release was observed in cells treated with eIF5A2 siRNA and doxorubicin compared with that in cells treated with doxorubicin alone, and eIF5A2 silencing accelerated doxorubicin-induced cell death (Fig. 4D). Notably, doxorubicin treatment alone activated apoptosis, but the apoptotic rate (early and late apoptosis) was only modestly further increased in the presence of eIF5A2 siRNA (Fig. 4E). These results suggested a limited additive effect between doxorubicin and eIF5A2 on apoptotic cell death.

Figure 4 Silencing of eIF5A2 induces sensitivity to doxorubicin in HCC cells by triggering lethal autophagy. (A) Transfection efficiency of sieIF5A2 in SNU449 cells. (B) SNU449 cells were infected with the mRFP-GFP-LC3 adenovirus, treated with Dox and sieIF5A2 alone or in combination, and visualized by confocal microscopy. The numbers of GFP+/mRFP+-LC3 (yellow, autophagosomes) and GFP−/mRFP+-LC3 (red, autolysosomes) dots were counted and analyzed. (C) Western blotting analysis of LC3, p62 and Beclin 1 proteins in SNU449 cells treated with Dox and sieIF5A2 alone or in combination. (D) LDH release was detected in SNU449 cells following the indicated treatments. Cell death of SNU449 cells was detected using trypan blue staining. (E) Apoptotic rates in SNU449 cells were assessed by annexin V-VITC/PI apoptosis assay using flow cytometry following the indicated treatments. Early and late apoptotic rates were analyzed. *P<0.05, **P<0.01 and ***P<0.001. eIF5A2, eukaryotic translation initiation factor 5A2; si, small interfering RNA; NC, negative control; mRFP, monomeric red fluorescent protein; GFP, green fluorescent protein; Dox, doxorubicin; LDH, lactate dehydrogenase.

eIF5A2 enhances HCC cell chemoresistance by suppressing autophagic cell death via a Beclin 1-dependent pathway

Since eIF5A2 siRNA and doxorubicin-induced autophagy was accompanied by cell death, we speculated that eIF5A2 knock-down may enhance doxorubicin-triggered autophagic cell death. To determine this, CQ, a pharmacological lysosomal inhibitor, was used to inhibit autophagy in SNU449 cells. Notably, treatment with CQ partially reversed doxorubicin sensitivity and diminished eIF5A2 siRNA-mediated doxorubicin sensitivity (Fig. 5A and B). In addition, CQ attenuated the doxorubicin-induced increase in LDH release and cell death (Fig. 5C and D). CQ also inhibited the LDH release and cell death induced by the combination of eIF5A2 siRNA and doxorubicin. By contrast, Rapa, which is an autophagy activator, significantly decreased doxorubicin-induced cell death and reversed the cytoprotective effects of the overexpression of eIF5A2 (Fig. 5E-H). These results suggested that eIF5A2 siRNA may enhance doxorubicin sensitivity by inducing autophagic cell death in HCC cells.

Figure 5 Combination of sieIF5A2 and Dox induces lethal autophagy. (A-D) SNU449 cells were treated with Dox, CQ and sieIF5A2 alone or in combination. (A) Transfection efficiency was assessed by western blotting. (B) Cell viability was assessed by CCK-8 assay. (C) LDH release was measured using an LDH assay. (D) Cell death was detected by Calcein-AM/PI staining. (E-H) SNU449 cells were treated with Dox, Rapa and OE eIF5A2 alone or in combination. (E) Transfection efficiency was assessed by western blotting. (F) Cell viability was assessed by CCK-8 assay. (G) LDH release was measured using an LDH assay. (H) Cell death was detected by Calcein-AM/PI staining. *P<0.05, **P<0.01 and ***P<0.001. eIF5A2, eukaryotic translation initiation factor 5A2; si, small interfering RNA; NC, negative control; CCK-8, Cell Counting Kit-8; LDH, lactate dehydrogenase; CQ, chloroquine; Rapa, rapamycin; OE, overexpression vector; Dox, doxorubicin.

To further investigate the underlying mechanisms, the protein levels of eIF5A2, LC-3 I/II, Beclin 1 and p62 in xeno-graft tumors were detected. Consistently with the results of the in vitro experiments, doxorubicin and eIF5A2 siRNA treatment increased the protein expression levels of LC3II and Beclin 1, but reduced the levels of p62 compared with those in the control group (Fig. 6A). Combination of doxorubicin and eIF5A2 siRNA induced higher LC3II and Beclin 1 and lower p62 protein expression levels compared with those observed following either therapy alone (Fig. 6A), indicating high levels autophagic activity in xenograft tumors under combined treatment. In addition, knockdown of Beclin 1 impaired the sensitivity of HCC cells to doxorubicin (Fig. 6B and C), and co-transfection with eIF5A2 siRNA did not restore the doxorubicin sensitivity of HCC cells following Beclin 1 knockdown (Fig. 6C). Similarly, knockdown of Beclin1 inhibited the combination treatment-induced LDH release and cell death (Fig. 6D and E). Taken together, these results demonstrated that the effects of eIF5A2 inhibition on autophagic cell death were mediated by Beclin 1.

Figure 6 eIF5A2 knockdown triggers cell death via a Beclin 1-dependent pathway. (A) Western blotting analysis of eIF5A2, LC3, p62 and Beclin 1 protein expression levels in hepatocellular carcinoma xenograft tumors dissected from mice at the end of the in vivo experiment. (B-E) SNU449 cells were treated with the indicated concentrations of Dox with single or double knockdown of eIF5A2 and Beclin 1. (B) Transfection efficiency was assessed by western blotting. (C) Cell viability was assessed by Cell Counting Kit-8 assay. (D) LDH release was measured using an LDH assay. (E) Cell death was detected by trypan blue exclusion assay. *P<0.05, **P<0.01 and ***P<0.001. eIF5A2, eukaryotic translation initiation factor 5A2; Dox, doxorubicin; si, small interfering RNA; NC, negative control; LDH, lactate dehydrogenase.

Discussion

HCC is the third leading cause of cancer-related death world-wide (2). Multi-drug resistance diminishes the efficiency of therapeutic agents in HCC; therefore, developing new strategies to enhance chemotherapy efficiency is urgent. The results of the present study demonstrated that the expression levels of eIF5A2 were significantly upregulated in tumor tissues compared with those in adjacent normal tissues and were negatively associated with the overall survival of patients with HCC. In addition, eIF5A2 levels were positively associated with doxorubicin resistance in HCC cells, and eIF5A2 silencing enhanced the antitumor effects of doxorubicin in vitro and in vivo. Further experiments revealed that eIF5A2 siRNA improved the therapeutic efficiency of doxorubicin in HCC by triggering autophagic cell death through a Beclin 1-dependent pathway.

eIF5A2 is an oncogene highly expressed in several types of human cancer, including melanoma, gastric, breast and gall-bladder cancer (11,12,34-36). Accumulating evidence suggests that eIF5A2 is involved in tumor initiation, growth, metastasis and chemoresistance (37-39). In particular, previous studies have demonstrated that inhibition or knockdown of eIF5A2 reverses chemoresistance in gastric and colorectal cancer, as well as in HCC (34,35,39-41). In addition, Yang et al (42) have reported that eIF5A2 overexpression significantly enhanced the resistance of esophageal squamous cell carcinoma cells to 5-fluorouracil by inhibiting apoptosis. Consistent with these, the present study demonstrated that eIF5A2 was upregulated in HCC, and eIF5A2 silencing enhanced the tumor-suppressing effects of doxorubicin both in vivo and in vitro. The results of the present study also demonstrated that overexpression of eIF5A2 induced resistance to doxorubicin. These results suggested a crucial role of eIF5A2 in the development of chemoresistance in HCC.

Autophagy is a double-edged sword, as the modulation (either activation or inhibition) of autophagy has been demonstrated to at least partly overcome or reverse doxorubicin resistance in breast cancer, hepatocellular carcinoma and osteosarcoma (43). Previous studies have suggested that autophagy serves as a pro-survival role, resulting in acquired resistance to chemotherapeutic agents in cancer (16,44). However, the cell death-promoting effects of autophagy have also been reported (25,45). For example, Tai et al (46) have demonstrated that sorafenib inhibits tumor growth by inducing autophagic cell death in HCC. Similarly, Wu et al (47) have reported that blocking β2 adrenoreceptor signaling enhanced sorafenib-induced autophagy and improved the antitumor effects of sorafenib in HCC. The results of the present study demonstrated that doxorubicin treatment induced autophagy, and that blocking autophagy with CQ impaired doxorubicin efficiency, suggesting that doxorubicin caused autophagic cell death. Notably, the results of further experiments revealed that knockdown of eIF5A2 enhanced cell death by triggering sustained lethal autophagy induced by doxorubicin. Beclin 1 was the first autophagy-related protein identified in mammals and serves a central role in autophagy (48). Beclin 1 constitutes a molecular platform for the regulation of autophagosome formation and maturation by forming distinct PI3K complexes together with the core lipid kinase VPS34 and the regulatory component VPS15 (49). Therefore, Beclin 1 can be used as a marker to monitor autophagy (17). The results of the present study demonstrated that knockdown of Beclin 1 reversed the eIF5A2 siRNA-mediated increase in doxorubicin sensitivity, revealing a potential mechanism of eIF5A2 siRNA-enhanced doxorubicin lethality.

In conclusion, the present study described the roles of eIF5A2 and autophagy in doxorubicin treatment of HCC. The results of the present study suggested that knockdown of eIF5A2 restored the sensitivity of HCC cells to doxorubicin by triggering lethal autophagy via a Beclin1-dependent signaling pathway. These results provide an potential target of HCC chemoresistance.

Supplementary Data

Acknowledgments

Not applicable.

Funding

This study was supported by the Zhejiang Provincial Ten Thousand Plan for Young Top Talents (2018), the Innovative Talents Training Project of Zhejiang Health Bureau (2018), the Key Project Co-constructed by the Zhejiang Province and Ministry (grant no. WKJ-ZJ-1916), the National High Technology Research and Development Program of China (grant no. SS2014AA020533), the Zhejiang Provincial Natural Science Foundation of China (grant no. LQ13H160006) and the Natural Science Foundation of China (grant nos. 81302071 and 81673809).

Availability of data and materials

The datasets generated and/or analyzed in the current study are available from the corresponding author on reasonable request.

Authors' contributions

YXT, WC and XNZ designed the study. KC and XRL performed the in vitro experiments and analyzed the data. JYZ, RRL, XXZ and SZX performed the in vivo experiments. HPK provided the experimental tools and analyzed the in vivo results. YXT, KC and XRL wrote the manuscript. WC and XNZ revised the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (Hangzhou, China), and written informed consent was obtained from all participants. All experimental procedures on animals were approved by the Institutional Animal Care and Use Committee at Zhejiang University School of Medicine.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References