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. 2025 May 10;34(11):2581–2589. doi: 10.1007/s10068-025-01890-3

A β-glucosidase extracted from walnut (Juglans regia L.) green husk induces human hepatoma cell apoptosis via ROS-mediated p53 and NF-κB pathway

Siyu Han 1, Lixia Zhao 3, Li Song 1,2,✉, Yuying Li 1,✉
PMCID: PMC12145352  PMID: 40492030

Abstract

Walnut green husk is the outermost green peel of walnut, which contains multiple natural ingredients with a variety of biological activities. In this study, an anti-tumor protein called Juglans regia L. husk protein (JRLHP) was isolated. Mass spectrometry and enzymatic activity analysis revealed that JRLHP was a β-glucosidase. JRLHP inhibited human hepatoma cancer cell growth and promoted apoptosis. JRLHP caused the decline of mitochondrial membrane potential and Bcl-2. Moreover, cytochrome c and Bax were elevated, and caspase-9 and caspase-3 were activated. JRLHP treatment led to excess reactive oxygen species (ROS). This was accompanied by increased p53 protein levels and the suppression of NF-κB pathway. NAC treatment reversed the effects of JRLHP on p53 and NF-κB pathway. Consequently, NAC also reduced the impacts of JRLHP on cell apoptosis and proteins related to apoptosis. Taken together, above results indicated that JRLHP induced HCC cell apoptosis by ROS-mediated p53 and NF-κB pathway.

Keywords: Walnut green husk, Apoptosis, ROS, p53, NF-κB pathway

Introduction

Hepatocellular carcinoma (HCC) is a malignant disease in the world. Incidence of liver cancer ranked the sixth of cancer (Bray et al., 2024). The situation of liver cancer is not encouraging due to the absence of early diagnosis and effective treatment. Usually, liver cancer patients will be treated with surgical resection, radiotherapy and chemotherapy, but its adverse prognosis and side effects can not be underestimated (Kim et al., 2018). At the same time, what is worrying is the recurrence rate of tumor (Filgueira, 2019). Therefore, exploring new and safer anticancer drugs is the focus of research in the field of cancer medicine.

Proteins extracted from plants have received widespread attention because of their convenient sources, rich types and small side effects. The green husk of walnuts is the byproduct of walnut fruit (Juglans regia L.) and discarded as waste after harvest. Studies showed that extracts from green husk have anti-tumor ability (Soto-Madrid et al., 2021). Juglanin and Juglone from walnut can induce cancer cell apoptosis (Avci et al., 2016; Sun et al., 2017). The green husk of walnuts has the potential to be a useful source of anti-tumor drugs.

Activating cancer cell apoptosis is a good strategy for cancer treatment. Many studies revealed that compounds from plants inhibited cancer cells by inducing apoptosis (Jiang et al., 2015; Song et al., 2016). The tumor suppressor protein p53 can regulate cell apoptosis by mitochondrial apoptotic pathways and promote the release of cytochrome C (Haupt et al., 2003; Taniguchi and Karin, 2018). Furthermore, apoptosis is induced by p53 mediating Bcl-2 family members (Dashzeveg and Yoshida, 2015). The nuclear factor-κB (NF-κB) signaling regulates apoptosis. Persistent activation of NF-κB signaling inhibits apoptosis (Taniguchi and Karin, 2018). NF-κB transcriptionally regulates anti-apoptotic proteins (Bours et al., 2000).

In the present study, an anticancer protein JRLHP was purified from walnut (Juglans regia L.) green husk. JRLHP hampered cell proliferation and caused apoptosis in HCC cells. Further investigation revealed that JRLHP caused apoptosis through mitochondrial-depended pathway, which was regulated by ROS-mediated p53 and NF-κB pathways.

Materials and methods

Purification and identification of JRLHP

The dried walnut green peel was immersed in a PBS buffer solution and left for an overnight soak at 4 °C. On the following day, filtration was utilized to remove the filter residue and the supernatant was collected twice. The crude protein in the supernatant was precipitated by acetone precipitation method. The residual acetone was evaporated, and the precipitation was suspended again with an appropriate amount of Tris buffer solution to obtain the crude protein extract. In order to remove pigments, activated carbon was joined and stirred continuously for 5 min. After filtration, purified protein was obtained and named as Juglans regia L. husk protein (JRLHP).

Purified JRLHP protein was examined via 12% SDS-PAGE. The strips were cut off and performed LC-MS/MS assay at Sangon Biotech (Shanghai, China).

β-Glucosidase activity assay of JRLHP

p-Nitrophenyl-β-D-glucopyranoside (p-NPG) was employed as the substrate to determine enzyme activity. The 2 mL reaction mixture containing 100 μL enzyme solution, 50 mM citric acid Na2HPO4 buffer (pH 6.0) and 0.5 mM p-NPG was incubated at 50 °C for 10 min. 1 mL 0.1 M Na2CO3 was added immediately to terminate the reaction. Absorbance at 410 nm was examined by a spectrophotometer. One unit of enzyme activity is defined as the quantity of enzyme that produces 1 μM of p-nitrophenol (PNP) from the substrate each minute.

Cell culture

Human hepatoma cell line Bel-7402, HepG2 and HL7702, obtained from the Institute of Cell Research (Shanghai, China), were maintained under standard culture conditions. Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All cultures were maintained in a humidified incubator at 37 °C with 5% CO2.

Cell viability assay

Cells were cultured in 96-well plates in a CO2 incubator overnight. The crude protein extract or JRLHP protein was added into the 96-well plates, followed by treating for 24 h. After sucking off culture medium, 20 μL of MTT solution was applied to each well and left for four hours. After sucking off the MTT solution, 150 μL of DMSO was added. The absorbance value was measured at 570 nm with a microplate analyzer.

Cell clone formation assay

Cells were cultured in a 12-well plate overnight and then treatedwith 20 μg/mL JRLHP for 7 days. The fresh medium containing JRLHP protein was replaced every 3 days. 0.1% crystal violet was used to dye cells after fixing. The 12-well plate was dried, and the number of cell clones was observed using a stereomicroscope. The rate of clone formation was calculated according to (number of clones/number of inoculated cells) × 100%.

Cell apoptosis assay

Cells were spread in a 12-well plate at 37 °C overnight, followed by treating with JRLHP protein for 24 h. After washing, cells were stained for ten minutes with DAPI. Then, the nucleus was observed using a microscope. The number of cells with apoptotic bodies was used to compute the apoptosis rate. The apoptosis was also determined by flow cytometry. Treated cells were digested and centrifugated. The cell precipitation was reconstituted using a solution that contained 2.5 µL of PI and 5 µL of Annexin V-FITC. Lastly, a FAC Sort flow cytometry was used to examine cell apoptosis.

Western blotting assay

Treated cells were collected. Radio immunoprecipitation assay (RIPA) buffer, cytoplasmic protein extraction kit, and nuclear protein extraction kit were used to isolate total, cytoplasmic, and nuclear proteins, respectively. The proteins are quantified, isolated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked and incubated for the entire night at 4 °C with the primary antibody. The next day, the primary antibody was removed. After adding the secondary antibodies, the mixture was incubated for one hour. Lastly, blots were visualized using chemiluminescence. The antibodies were used including antibodies against Cytochrome c (1:1000, BBI), Bcl-2 (1:500, BBI), Bax (1:500, BBI), activated-caspase-3 (1:500, Beyotime Biotechnology), activated-caspase-9 (1:500, Abcam), p53 (1:1000, Abcam), p65 (1:500, Beyotime Biotechnology), laminB (1:500, Bioworld), α-tubulin (1:500, Sigma-Aldrich).

ROS level detection

Treated cells were incubated with 10 μM of 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) for 30 min. After discarding the supernatant, cells were digested, collected and resuspended. Fluorescence changes induced by DCF accumulation were detected by a fluorescence microplate reader (Thermo Scientific Varioskan Flash).

Statistical analysis

Statistical analysis was performed using SPSS Statistics 22. Data were presented as mean ± standard deviation. One-way analysis of variance was used to determine the significance between the groups. Among them, p < 0.05 indicated statistical significance.

Results and discussion

Purification and identification of JRLHP

The crude protein was isolated from walnut green husk. MTT assay was performed to evaluate its effect on human hepatoma cell line Bel-7402. The results indicated that the crude protein significantly reduced the survival rate of Bel-7402 cells (Fig. 1A). SDS-PAGE analysis indicated that crude extraction from walnut green husk contains a single band protein (Fig. 1B). However, we found crude extraction contained more pigments. So, we further purified antitumor active protein by removing the pigment of crude extraction using activated carbon adsorption. Finally, a pure protein named as Juglans regia L. husk protein (JRLHP) was obtained (Fig. 1B). The yield of JRLHP was 21.62 mg/100 g dry walnut green peel (Table 1). JRLHP was further identified by LC–MS/MS. The results indicated 71 peptides were matched to beta-glucosidase 24-like (XP_035541426.1) of Juglans regia, with a Mascot probability score of 1216 (Fig. 1C). The sequence coverage is 43% (Fig. 1C). Beta-glucosidase are glycoside hydrolase that catalyze the hydrolysis of glycosidic bonds (Ketudat Cairns and Esen, 2010). So, the beta-glucosidase activity of JRLHP was analyzed. The results showed JRLHP is a beta-glucosidase (Table 1).

Fig. 1.

Fig. 1

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Purification of JRLHP from walnut green husk. (A) Effects of crude extraction on Bel-7402 cell viability. The data are from three separate experiments and are shown as mean ± standard deviation. ** represents a significant difference compared to the control group (p < 0.01). (B) SDS-PAGE analysis. Line M: protein marker; Line 1: crude extraction; Line 2: protein purified by activated carbon decolorization. (C) The sequence of beta-glucosidase 24-like (XP_035541426.1). Matched peptides shown in Red

Table 1.

Purification steps of JRLHP from walnut green husk

Purification steps Total protein (mg) Total activity (U) Specific activity (U/mg) Fold Recovery (%) IC50 (µg/mL)
Crude extraction 27.34 ± 11.58 30,310.0 ± 532.9 1108.63 ± 1.3 1.00 100 88.95 ± 3.07
Activated carbon decolorization 21.62 ± 10.91 24,634.48 ± 358.0 1139.43 ± 4.5 1.03 81.3 64.01 ± 2.21
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JRLHP inhibited human hepatoma cell proliferation

The anti-tumor acitivities of JRLHP were further analyzed by detecting its impacts on the proliferation of hepatoma cells (Bel-7402 and HepG2) and normal hepatocytes (HL7702) using MTT assay. MTT results revealed that JRLHP inhibited cell viabilities of HCC cells, and had no obvious inhibitory effects on HL7702 cells (Fig. 2A). Bel-7402 and HepG2 cells exhibited IC50 values of 64.01 ± 2.21 μg/mL and 60.5 ± 3.82 μg/mL, respectively. Additionally, a clonogenic survival assay was employed, revealing that JRLHP substantially decreased the clone formation rate of Bel-7402 and HepG2 cells (Fig. 2B, C). The above results show that JRLHP extracted from walnut green husk can effectively inhibit the proliferation of HCC cells. JRLHP is a beta-glucosidase. At present, there are no reports about the direct inhibitory effects of beta-glucosidase on tumor. We further investigated the effects of a β-glucosidase from almonds on Bel-7402 and HepG2 cell viability. We found that 45 and 60 µg/mL β-glucosidase from almonds significantly inhibited the Bel-7402 and HepG2 cell viability (Fig. 2D). These results indicated that β-glucosidases may have the same anti-cancer effects.

Fig. 2.

Fig. 2

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JRLHP inhibited human hepatoma cell proliferation. (A) Cell viability assay. (B) The assay for colony formation. (C) The rate of colony formation. (D) Effects of β-glucosidase from almonds on Bel-7402 and HepG2 cell viability. In (A), (C) and (D), data are percent as the mean ± SD of three independent experiments. **p < 0.01 in comparison to the control group

JRLHP induced HCC cell apoptosis by the mitochondria pathway

To determine if JRLHP’s inhibitory effect on HCC cell proliferation is associated with apoptosis, HCC cells were treated with JRLHP for 24 h. Subsequently, we employed DAPI staining and flow cytometry to assess cell apoptosis. Figure 3A showed HCC cells treated with JRLHP exhibited the apoptotic characteristics with typical apoptotic bodies. With the increase of protein concentration, the number of apoptotic bodies increased (Fig. 3A). Meanwhile, flow cytometry assay showed that he cell apoptosis trend gradually increased with the increase of JRLHP concentrations (Fig. 3B).

Fig. 3.

Fig. 3

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JRLHP promoted HCC cell apoptosis through the mitochondrial pathway. (A) Images of cell nuclei stained with DAPI and apoptosis ratio from DAPI staining. (B) Flow cytometry analysis and apoptosis ratio form flow cytometry. (C) Images of cells stained with JC-1 and the ratio of red/green fluorescence. (D) Western blotting assay of apoptosis-related proteins. (E) Relative protein levels according to western blotting assay. In (A), (B), (C) and (E), **p < 0.01 in contrast to the control group

Mitochondria play a crucial role in apoptosis induction and regulation (Sinha et al., 2013). A key indicator of early apoptosis is the loss of mitochondrial membrane potential (∆ψm). In order to investigate whether JRLHP-induced apoptosis is through the mitochondrial pathway, JC-1was used to detect ∆ψm in JRLHP-treated cells. In healthy cells, JC-1 accumulates in mitochondria, emitting red fluorescence. JC-1 staining showed that JRLHP treatment reduced ∆ψm of HCC cells (Fig. 3C). Many studied found that a series of proteins are involved in the regulation of mitochondrial apoptosis pathways. The Bcl-2 family proteins, comprising pro- and anti-apoptotic members, govern the mitochondrial apoptotic pathway (Martinou and Green, 2001). Cell fate is determined by the balance between pro-apoptotic proteins (e.g., Bax, Bid, Bim) and anti-apoptotic proteins (e.g., Bcl-2, Bcl-xL, Mcl-1) (Czabotar et al., 2014). Bax is inactive when binding with Bcl-2. Upon receiving death signal, Bcl-2 is inhibited and Bax is activated. This leads to cytochrome c release and activation of caspases-3 and -9, promoting apoptosis. So, the effects of JRLHP on mitochondria pathway-related proteins were analyzed. Results demonstrated significant increases in cytochrome c, Bax, active-caspase-9, and active-caspase-3 protein levels, while the anti-apoptotic Bcl-2 protein level decreased markedly (Fig. 3D, E). These findings indicated that JRLHP triggered HCC cell apoptosis via the mitochondria-mediated pathway.

JRLHP regulates p53 and NF-κB pathway through ROS

Tumor suppressor protein p53 and NF-κB pathway play crucial roles in tumorigenesis (Finlay et al., 1989; Yu et al., 2020). Studies found that p53 and NF-κB signaling could regulate mitochondrial apoptotic pathway (Haupt et al., 2003; Taniguchi and Karin, 2018). To seek the mechanism of JRLHP inducing cell apoptosis, we investigated the effects of JRLHP on p53 and NF-κB signaling. As shown in Fig. 4A, p53 protein levels were unregulated significantly in JRLHP-treated HCC cells. Meanwhile, total, nuclear and cytoplasmic p65 protein levels decreased significantly after treatment with JRLHP (Fig. 4B, C). Both p53 and NF-κB pathway was reported to regulate apoptosis via Bcl-2 family (Bours et al., 2000; Dashzeveg and Yoshida, 2015). P53 negatively mediated Bcl-2 protein expression at the post-translational level and transcriptionally promotes Bax expressions (Dashzeveg and Yoshida, 2015; Miyashita et al., 1994). P53 also binds to Bcl-2, which triggers mitochondrial outer membrane permeabilization (MOMP) and cytochrome C release (Tomita et al., 2006). P65 is one of the NF-κB’s five family members (Hoesel and Schmid, 2013). P65 enters cell nuclear and then inhibits apoptosis by the transcription of anti-apoptotic proteins (Bours et al., 2000). Our results showed JRLHP treatment led to upregulation of p53 and downregulation of p65, but also led to downregulation of Bcl-2 protein and upregulation of Bax. These results suggested JRLHP induced apoptosis by regulating Bcl-2 and Bax, which was mediated by p53 and NF-κB pathways.

Fig. 4.

Fig. 4

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JRLHP regulated p53 and NF-κB pathways through ROS. (A) Western blotting assay of p53 expression in JRLHP-treated HCC cells. (B) Western blotting assay of p65 in JRLHP-treated HCC cells. (C) Relative protein levels of p65. (D) ROS levels in JRLHP-treated HCC cells. In (A), (C) and (D), **p < 0.01 in comparison with control group. (E) Impacts of NAC pretreatment on ROS levels in JRLHP-treated HCC cells. (F) Western blotting was employed to detect the effects of NAC pretreatment on the expressions of p53 and p65 protein in JRLHP-treated HCC cells. (G) Relative protein levels of p65 and p53 proteins. In (E) and (G), **p < 0.01 compared to cells treated with JRLHP alone

Increasing reactive oxygen species (ROS) generation is an effective strategy for cancer treatment (Mendes et al., 2022). We detected the influence of JRLHP on ROS. The results showed that JRLHP treatment increased the accumulation of ROS (Fig. 4D). ROS is closely related to both p53 and NF-κB pathway. ROS can promote the activation of p53, which leads to cell apoptosis (Callen, 2013). Intracellular ROS also could inhibit the NF-κB pathway, which then promotes cell apoptosis (Morgan and Liu, 2011). We found NAC (ROS scavenger) pretreatment could decrease ROS levels and antagonized the effects of JRLHP on p53 and NF-κB pathway (Fig. 4E–G). These results suggested that JRLHP induced p53 activation and NF-κB pathway inhibition by ROS.

JRLHP induced HCC cell apoptosis through ROS

To further examine the involvement of ROS in JRLHP-triggered cell apoptosis, we detected the effects of NAC on the survival inhibition and apoptosis of HCC cells induced by JRLHP. As illustrated in Fig. 5A, the apoptosis rate was notably reduced in the group treated with both NAC and JRLHP compared to the group treated with JRLHP alone. Similarly, the survival rate was increased significantly after NAC pretreatment (Fig. 5B). Furthermore, the effects of NAC on apoptosis-related proteins were measured. We discovered that NAC pretreatment reversed the changes of apoptosis-related proteins in the JRLHP treatment group (Fig. 5C, D). JRLHP induced p53 activation and NF-κB pathway inhibition by ROS. Excessive ROS was known to induce mitochondria-dependent apoptosis (Sastre et al., 2000). In line with this, NAC treatment inhibited JRLHP-induced apoptosis. Moreover, our study found that JRLHP regulated p53 activation and NF-κB pathway inhibition through ROS. These results suggested that JRLHP induced HCC cell apoptosis by ROS-mediated p53 and NF-κB pathway.

Fig. 5.

Fig. 5

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Effects of NAC pretreatment on cell apoptosis, cell viability and apoptosis-related proteins in JRLHP-treated HCC cells. (A) The effects of NAC pretreatment on JRLHP-induced apoptosis of HCC cells were detected by flow cytometry. (B) Effects of NAC pretreatment on cell viability of JRLHP-treated HCC cells. (D) Western blotting was used to detect the effects of NAC pretreatment on apoptosis-related proteins in JRLHP-treated HCC cells. (E) Relative protein levels. In (A), (B) and (D), **p < 0.01 compared to cells treated with JRLHP alone

Acknowledgements

This study was supported by Natural Sciences Foundation of Shanxi (20210302123462), Open Fund Project of Xinghuacun College of Shanxi University (XCSXU-KF-202321), Scientific Research Project in Traditional Chinese Medicine of Shanxi Province (2024ZYYB053) and Key Research and Development Project of Xinzhou Science and Technology Plan (No. 20240202).

Declarations

Conflict of interest

No potential conflicts of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Li Song, Email: lsong@sxu.edu.cn.

Yuying Li, Email: lyy9030@sxu.edu.cn.

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