Chloride intracellular channel 6 inhibits hepatocellular carcinoma progression by modulating immune cell balance and promoting tumor cell apoptosis

Establishment of liver cancer animal model

Thirty male BALB/c nude mice, aged 6–8 weeks and weighing 25 ± 2 g, were purchased from the Institute of Materia Medica, Chinese Academy of Sciences. This study was approved by the Experimental Animal Welfare Ethics Committee of the Association. In accordance with the random number table method, the nude mice were divided into control group, model group, model + low CLIC6 (10 nM/day, ab106875, Abcam, Cambridge, MA, USA) group, model + medium CLIC6 (20 nM/day) group, and model + high CLIC6 (50 nM/day) group.^[12] Pentobarbital sodium (40 mg/kg, 21642-83-1, Shandong Xiya Chemical Industry Co., Ltd., Shandong, China) was injected intraperitoneally to anesthetize the nude mice. Using a 1 mL sterile syringe, an appropriate amount of HepG2 cell (iCell-h092, iCELL, Shanghai, China) suspension (approximately 100 μL, containing 2 × 10⁶ cells) was slowly injected subcutaneously into the back of the nude mice to establish a subcutaneous xenograft liver cancer model. Caution was taken to ensure that the cell suspension was evenly distributed without forming bubbles. After the injection, the nude mice were returned to sterile cages, and their health status was monitored. Tumor growth was regularly observed. Different doses of CLIC6 were administered to the model + low CLIC6 (10 nM/day) group, model + medium CLIC6 (20 nM/day) group, and model + high CLIC6 (50 nM/day) group via tail vein injection on the basis of the model group. A daily injection of an equivalent volume of saline was administered to the control group. The nude mice were sacrificed using an intraperitoneal dose of pentobarbital sodium (110 mg/kg) following a 21-day experiment. Tumor and liver tissues were collected for further analysis.

ELISA assay

The serum levels of mice were analyzed with NO (S0021S), malondialdehyde (MDA, S0131S), and super oxide dismutase (SOD, S0101S) ELISA kits. Kits purchased from Beyotime Biotechnology (Shanghai, China). Perform experiments according to kit instructions. The analysis was carried out by enzyme-labeled apparatus (Multiskan SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA).

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining

Tissue samples were embedded in paraffin. The slides were deparaffinized by immersing them in xylene (2 times, 5 min each, 534056, Merck, Darmstadt, Germany) and then rehydrated through a series of ethanol solutions: 100%, 95%, 85%, and 70% (2 min each). Finally, they were rinsed with distilled water. The sections were incubated with proteinase K solution (20 μg/mL in phosphate buffer saline (PBS), 10412ES, Yeasen, Shanghai, China) for 15–30 min at room temperature and then rinsed with PBS. The sections were treated with 0.1% Triton X-100 (P0096, Beyotime Biotechnology, Shanghai, China) in PBS for 5–10 min at room temperature and then rinsed with PBS. A TUNEL reaction (C1091, Beyotime Biotechnology, Shanghai, China) mixture was prepared in accordance with the manufacturer’s instructions, usually containing terminal deoxynucleotidyl transferase (TdT) enzyme and biotinylated nucleotide mix. This mixture was applied to the sections and incubated in a humidified chamber at 37°C for 1 h. The sections were rinsed with PBS to stop the reaction, incubated with streptavidin- horseradish peroxidase ( HRP) conjugate for 30 min at room temperature, and rinsed with PBS. The sections were mounted with an appropriate mounting medium and covered with coverslips. They were observed under a light microscope (CX53, Olympus, Tokyo, Japan).

Immunohistochemistry

First, tissue samples were embedded in paraffin, or sections were prepared. Then, the slides were immersed in xylene to deparaffinize (2 times, 5 min each), rehydrated through a series of ethanol solutions (100%, 95%, 85%, and 70%, 2 min each), and rinsed with distilled water. Next, the sections were heated in citrate buffer (pH 6.0, C999, Sigma, St. Louis, MO, USA) or ethylene diamine tetraacetic acid (EDTA) buffer (pH 9.0, 4008M, Sigma, St. Louis, MO, USA) for 10–20 min for antigen retrieval, cooled to room temperature, and rinsed with PBS. The sections were rinsed with PBS again after 10 min of immersion in 3% H₂O₂ at room temperature to inhibit endogenous peroxidase activity. Then, the sections were incubated in normal serum (such as 10% goat serum or horse serum) for 30 min to block nonspecific binding sites. Subsequently, the sections were incubated with specific primary antibodies for phosph-JNK (1:500, ab307802), T-box expressed in T cell (T-bet) (1:500, ab307193), or cleaved caspase-3 (1:500, ab32042), typically overnight at 4°C or for 1–2 h at room temperature, and then rinsed with PBS. Next, the sections were incubated with secondary antibodies (1:1000, ab6721) for 30 min at room temperature and rinsed with PBS. All antibodies were obtained from Abcam (Cambridge, MA, USA). A 3,3’-diaminobenzidine ( DAB) substrate kit (ml095670, Shanghai Enzyme-linked Biotechnology Co., Ltd., Shanghai, China) was used for color development until a brown color appeared (usually 5–10 min) and rinsed with tap water to stop the reaction. The sections were counterstained with hematoxylin (C0107, Beyotime Biotechnology, Shanghai, China) for 1–2 min to stain the nuclei blue and rinsed with tap water. Finally, the sections were mounted with an appropriate mounting medium and covered with coverslips. They were observed under a light microscope (CX53, Olympus, Tokyo, Japan). The images were analyzed by ImageJ (version 1.3.4, National Institutes of Health, Bethesda, MD, USA), and the results were calculated in accordance with integrated optical density (IOD)/area.

RNA extraction and quantitative real-time polymerase chain reaction (PCR)

Total ribonucleic acid ( RNA) was extracted from tissue samples using a TRIzol universal reagent (DP424, TIANGEN, Beijing, China). Subsequently, the RNA was transcribed into complementary DNA (cDNA) using reverse transcriptase (KR103, TIANGEN, Beijing, China), with the addition of specific primers to enhance cDNA synthesis efficiency. Specific primer pairs were designed, with one serving as the forward primer and the other as the reverse primer for subsequent PCR amplification of the cDNA. The prepared PCR reaction mixture included the cDNA template, primers, DNA polymerase, and other necessary reagents and involved cycles of denaturation, annealing, and extension. Real-time monitoring of fluorescence signals during PCR reflected proportional amplification of PCR products. Data analysis involved quantifying target gene expression levels using standard curves or the 2^−ΔΔCt method. Finally, the obtained data were compared and interpreted relative to the reference gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) to assess gene expression differences between samples. The primer sequences employed are detailed in Supplementary Table 1.

Western blot

First, the protein in the sample was extracted with radioimmunoprecipitation assay ( RIPA, P0013B, Beyotime Biotechnology, Shanghai, China), and its concentration was measured with a bicinchoninic acid assay kit (P0011, Beyotime Biotechnology, Shanghai, China) to ensure quality. Next, the proteins were separated by size through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred onto polyvinylidene fluoride (IPVH00010, Millipore Corporation, MA, USA) or nitrocellulose membrane. The membrane was then incubated with specific primary antibodies phosph- signal transducer and activator of transcription (p-STAT) 3 (1:1000, ab267373), signal transducer and activator of transcription (STAT) 3 (1:1000, ab 68153), p-p38 (1:1000, ab195049), p38 (1:1000, ab170099), phospho-janus kinase (p-JAK) 1 (1:1000, ab138005), Janus kinase (JAK) 1 (1:1000, ab133666), CLIC6 (1:1000, PA5-101519, Thermo Fisher Scientific, Waltham, MA, USA), and GAPDH (1:1000, ab9485) washed with buffer after binding the target protein. All primary antibodies were obtained from Abcam (Cambridge, MA, USA). Subsequently, a secondary antibody labeled with an enzyme or fluorescent substance was applied, followed by another washing with buffer to remove unbound secondary antibodies (1:2000 dilution; cat nos. ZB-2305, ZB-2301, ZSGB-BIO, Beijing, China). Finally, gel imaging systems were used to capture the staining signals for analysis. Typically, results are compared to a reference protein (GAPDH) to ensure data accuracy and reliability. An enhanced chemiluminescence kit (BL520b, Biosharp Life Science, Hefei, Anhui, China) was used to develop the protein, and ImageJ (version 1.3.4, National Institutes of Health, Bethesda, MD, USA) was used to analyze the gray values of protein bands.

Cell culture

HepG2 cells (iCell-h092) were purchased from iCell (Shanghai, China). Dulbecco’s modified eagle medium (DMEM) was supplemented with 10% fetal bovine serum (C0226, Beyotime Biotechnology, Shanghai, China) and 1% penicillin-streptomycin solution (C0222, Beyotime Biotechnology, Shanghai, China). The frozen HepG2 cells were thawed, transferred to a centrifuge tube containing the medium, and centrifuged. The supernatant was discarded, and the cells were resuspended and cultured at 37°C with 5% carbon dioxide. When the cell confluence reached 80–90%, the cells were passaged, washed with PBS, and incubated with trypsin-EDTA solution. The cells were added with medium to neutralize the trypsin, centrifuged, resuspended, and passaged at a 1:3 or 1:5 ratio. For cell cryopreservation, the cells were resuspended in a freezing solution containing 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO), aliquoted into cryovials, placed in a −80°C freezer overnight, and transferred to a liquid nitrogen tank for long-term storage. The cells used in this study underwent short tandem repeat (STR) authentication and were tested negative for mycoplasma.

5-Ethynyl-2'-deoxyuridine (EdU) staining of HepG2 cells

First, HepG2 cells were cultured in a 96-well plate until the cell confluence reached 70–80%. Subsequently, EdU solution (10 μM, E-CK-A377, Elabscience, Wuhan, China) was added to each well in accordance with the experimental design, and the wells were incubated for 1–2 h to promote cellular uptake. After the cells were fixed with 4% paraformaldehyde, they were washed with PBS. Next, a 0.5% Triton X-100 solution was used to permeabilize the cells, and the cells were given another PBS wash. EdU staining reaction (C0085S, Beyotime Biotechnology, Shanghai, China) was implemented using a click chemistry reaction kit, and the cells were incubated at room temperature in the dark for 30 min. After being stained, the cells were washed with PBS and optionally stained with 4',6-diamidino-2'-phenylindole (DAPI) to label the cell nuclei. Finally, the images of EdU-positive cells were observed and captured using a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

TUNEL staining of HepG2 cells

First, HepG2 cells were fixed with 4% paraformaldehyde to preserve their morphology. Subsequently, the cells were permeabilized using a permeabilization agent, such as 0.1% Triton X-100, to allow the TUNEL reaction mixture to enter the cells. Next, a reaction mixture containing TdT enzyme and fluorescently labeled dUTP was added to the samples, typically incubating at room temperature for 30 min. The reaction was terminated by adding a stop reaction solution. Finally, the cell nuclei were optionally stained with DAPI, and the results were observed and analyzed using a fluorescence microscope (BX53, Olympus, Tokyo, Japan).

Statistical analyses

The experimental data were statistically analyzed to assess significant differences between conditions and ensure data reliability. Each experiment was replicated at least 3 times, and the results are presented as the mean ± standard error of the mean. Data processing and visualization were performed using Statistical Package for the Social Sciences software (version 17.0, IBM Corp., Armonk, NY, USA). Analysis of variance was used for intergroup comparisons, with Tukey’s post hoc test or Bonferroni correction applied on the basis of experimental design. P < 0.05 was considered statistically significant.

CLIC6 inhibited the growth and development of liver cancer tumor tissues

A subcutaneous tumor-bearing mice model was established, and tumors were obtained, as shown in Figure 1. The results in Figure 1a-c indicate that the tumor size, volume, and weight of the model group were significantly larger than those of the high-, medium-, and low-dose CLIC6 treatment groups (P < 0.001). The tumor size, volume, and weight gradually decreased with increasing doses of CLIC6, with the high-dose CLIC6 treatment group showing the most pronounced therapeutic effect (P < 0.001). The hematoxylineosin staining results [Figure 1d] showed that compared with the model group, the tumor tissues of the CLIC6 treatment groups with different doses exhibited varying degrees of damage, increased gap, and disorganized cell arrangement, with the high-dose CLIC6 treatment group demonstrating the most significant effect (arrows are shown in Figure 1).

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Figure 1:

CLIC6 suppressed the growth and development of liver cancer tumor tissues. (a) Representative images of liver cancer tumors after treatment with different doses of CLIC6. (b) Tumor volume. (c) Tumor weight. (d) Tumor tissues examined using HE staining following varying dosages of CLIC6 intervention, objectve: 200×. (n = 3). Scale bar: 100 μm. n = 6 (^✶✶P < 0.01 and ^✶✶✶P < 0.001). CLIC6: Chloride intracellular channel 6, HE: Hematoxylin–eosin.

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CLIC6 promoted the apoptosis of HCC cells

In Figures 2a and b, the expression of cleaved caspase-3 protein increased significantly at different doses of CLIC6 (P < 0.001). CLIC6 treatment markedly increased the Bax mRNA level and lowered the Bcl-2 mRNA level (P < 0.01 and P < 0.001; [Figure 2c]). The high dose of CLIC6 (20 nM) showed a more significant therapeutic effect than the low and medium doses. The TUNEL staining results [Figure 2d and e] showed that the apoptosis level of tumor tissue treated with high, medium, and low doses of CLIC6 significantly increased (P < 0.05 and P < 0.001, respectively). Among them, the CLIC6 (20 nM) treatment group showed the highest level of apoptosis (P < 0.001).

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Figure 2:

CLIC6 enhanced apoptosis of hepatocellular carcinoma cells. (a and b) Immunohistochemistry staining of cleaved caspase-3. Scale bar: 100 μm. (c) mRNA levels of Bcl-2 and Bax in tumor tissues. (d and e) TUNEL staining, objectve: 200×, detection of apoptosis level in mice liver cancer tumor tissues. Scale bar: 100 μm. n = 3 (ns: No significant difference; ^✶P < 0.05, ^✶✶P < 0.01, and ^✶✶✶P < 0.001). CLIC6: Chloride intracellular channel 6, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, Bcl-2: B-cell lymphoma-2, BAX: Bcl-2-associated X, mRNA: messenger RibonucleicAcid.

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CLIC6 is involved in regulating inflammatory response and immune cell equilibrium

As shown in Figure 3a-c, the CLIC6 expression was measured at the molecular and mRNA levels, and it was positively correlated with the dose (P < 0.05 and P < 0.001, respectively). In Figure 3d-j, the levels of Interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), interferon (IFN)-γ, and IL-17A in the liver tissue of the model group were higher (P < 0.001). However, the mRNA levels of IL-4 in the 5 nM CLIC6 group decreased (P < 0.05). The mRNA levels of IL-1β, IL-6, TNF-α, IFN-γ, and IL-17A in the 5 nM CLIC6 group decreased (P < 0.05, P < 0.01, and P < 0.001). Meanwhile, these mRNA levels in the medium- and high-dose CLIC6 treatment groups (10 and 20 nM) significantly decreased, whereas the IL-4 and transforming growth factor-β (TGF-β) mRNA levels significantly increased (P < 0.001). In Figure 3k and l, the positive expression of T-bet in the model group was significantly high (P < 0.001). However, the T-bet expression was clearly reduced in all CLIC6 and positive drug treatment groups (P < 0.001). Figure 3m demonstrates that the ROR-γO expression was dramatically downregulated (P < 0.001) in the 20 nM CLIC6 group and significantly upregulated (P < 0.01) in the liver. The expression of ROR-γt was distinctly downregulated in the 20 nM CLIC6 group (P < 0.001). The expression levels of GATA-binding protein 3 (GATA3) and forkhead box protein P3 (Foxp3) in the model group significantly decreased (P < 0.001). The Foxp3 and GATA3 levels significantly increased after 20 nM of CLIC6 treatment (P < 0.001). In addition, CLIC6 can regulate the secretion of immune T cells.

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Figure 3:

CLIC6 is involved in inflammatory response and immune cell balance in liver tissue. (a-c) CLIC6 level in liver analyzed by Western blot and qRT-PCR. (d-j) mRNA levels of IL-1β, TNF-α, IFN-γ, IL-6, IL-17A, IL-4, and TGF-β in liver. (k and l) T-bet staining, objectve: 200×, expression in mice liver. Scale bar: 100 μm. (m) Levels of ROR-γt, Foxp3, and GATA3 in liver. n = 3 (ns: No significant difference; ^✶P < 0.05, ^✶✶P < 0.01, and ^✶✶✶P < 0.001). CLIC6: Chloride intracellular channel 6, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, IL: Interleukin, TNF: Tumor necrosis factor, IFN: Interferon, TGF: Transforming growth factor, T-bet: T-box expressed in T cell, ROR-γt: Retinoic acid receptor-related orphan receptor gamma-t, Foxp3: Forkhead box protein P3, GATA3: GATA-binding protein 3, qRT-PCR: Quantitative real-time polymerase chain reaction.

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CLIC6 influenced the progression of liver cancer by improving p-JNK-mediated oxidative stress

The liver tissue of the model group demonstrated considerably high p-JNK expression, as shown in Figures 4a and b. Different doses of CLIC6 significantly reduced the positive expression of p-JNK (P < 0.001). In Figures 4c and d, the phosphorylation expression levels of p38 and STAT3 significantly increased (P < 0.001). However, medium and high doses of CLIC6 significantly reduced the p-STAT3 and p-p38 protein levels in the mice liver (P < 0.001). As shown in Figure 4e-g, the serum MDA and NO levels in the model group significantly increased, whereas the SOD level significantly decreased (P < 0.001). The serum MDA and NO levels in the medium- and high-dose CLIC6 groups significantly decreased (P < 0.05), and the SOD level significantly increased (P < 0.01).

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Figure 4:

CLIC6 is involved in p-JNK-mediated oxidant stress. (a and b) Immunohistochemistry staining, objectve: 200×, of p-JNK. Scale bar: 100 μm. (c and d) Expression levels of p-STAT3 and p-p38 proteins in liver. (e-g) Serum levels of MDA (e), NO (f), and SOD(g). (h) IFN-β mRNA levels in liver. (i and j) Protein expression levels of JAK1 and p-JAK1. n = 3 (ns: No significant difference; ^✶P < 0.05, ^✶✶P < 0.01, and ^✶✶✶P < 0.001). CLIC6: Chloride intracellular channel 6, JAK: Janus tyrosine kinase, STAT: Signal transducer and activator of transcription, p38: P38 mitogen-activated protein kinase, IFN: Interferon, p-JNK: phosphor-c-Jun N-terminal kinase, MDA: Malondialdehyde, SOD: Super Oxide Dismutase.

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Meanwhile, the expression of IFN-β mRNA in the model group increased (P < 0.001). CLIC6 treatment at different doses significantly reduced the level of IFN-β mRNA (P < 0.05 [Figure 4h]). The p-JAK1 protein expression of the control group was noticeably high (P < 0.001 [Figure 4i and j]). The p-JAK1 protein levels in the CLIC6 groups were reduced (P < 0.01).

CLIC6 inhibited the proliferation of HepG2 cells and promoted apoptosis

Next, the role of CLIC6 in inhibiting liver cancer development was further confirmed at the cellular level in vivo. Figure 5a shows that 10 and 20 nM of CLIC6 significantly inhibited the activity of HepG2 cells in a dose-dependent manner (P < 0.001), with the highest dose of CLIC6 recombinant protein resulting in the lowest cell activity. In Figure 5b-d, with the increase in CLIC6 dose, the CLIC6 level in cells increased significantly (P < 0.05 and P < 0.001). As shown in Figures 5e and f, EdU staining showed a negative correlation between CLIC6 dose and cell proliferation. The TUNEL staining results [Figure 5g and h] indicated that treatment with CLIC6 recombinant protein significantly promoted apoptosis in HepG2 cells, with the high-dose CLIC6 group showing the most pronounced effect (P < 0.001).

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Figure 5:

CLIC6 suppressed proliferation of HepG2 cells and induced apoptosis. (a) Activity of HepG2 cells treated with different doses of CLIC6 recombinant protein. (b-d) Levels of CLIC6 in cells were analyzed by Western Blot and qRT-PCR. (e and f) EdU staining, objectve: 200×, was used to analyze the effects of different doses of CLIC6 recombinant protein on the proliferation capacity of HepG2 cells. Scale bar: 50 μm. (g and h) Cell apoptosis measured using TUNEL staining, objectve: 200×, after treating HepG2 cells with different doses of CLIC6 recombinant protein. Scale bar: 50 μm. n = 3 (ns: No significant difference; ^✶P < 0.05, ^✶✶P < 0.01, and ^✶✶✶P < 0.001). CLIC6: Chloride intracellular channel 6, EdU: 5-ethynyl-2'-deoxyuridine, DAPI: 4',6-diamidino-2'-phenylindole, qRT-PCR: Quantitative real-time polymerase chain reaction, TUNEL: Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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